1. Field of the Invention
The present invention relates to a linker for optimizing a unimolecular FRET biosensor based on the principle of fluorescence resonance energy transfer; a biosensor containing the linker; a gene encoding for the linker or the unimolecular FRET biosensor; an expression vector containing the linker or the unimolecular FRET biosensor; a transformed cell and a transgenic non-human animal harboring the expression vector; and a measurement method using the biosensor.
2. Description of the Related Art
Biosensors utilizing the principle of fluorescence resonance energy transfer (hereinafter may be referred to as “FRET”) and fluorescent proteins have been increasingly used in the field of life science (see Aoki, K. and M. Matsuda. 2009. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nature Protocol 4:1623-1631, Giepmans, B. N., S. R. Adams, M. H. Ellisman, and R. Y. Tsien. 2006. The fluorescent toolbox for assessing protein location and function. Science 312:217-224, Jares-Erijman, E. A. and T. M. Jovin. Imaging molecular interactions in living cells by FRET microscopy. 2006. Curr. Opin. Chem. Biol. 10:409-416, and Kiyokawa, E., S. Hara, T. Nakamura, and M. Matsuda. 2006. Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells. Cancer Sci. 97:8-15).
The FRET is a phenomenon of excitation energy transfer from an excited fluorescent molecule (donor: energy donor) to a fluorescent molecules in the close vicinity of the donor (acceptor: energy acceptor). A development and improvement of Green fluorescent protein (GFP) mutants of different colors have greatly contributed to the spread of the biosensors utilizing the FRET. Nowadays, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), which both are GFP mutants, have often been used as donor and acceptor fluorescent proteins.
FRET biosensor systems utilizing the fluorescent proteins are classified into two types: bimolecular FRET biosensors (see FIG. 1) and unimolecular FRET biosensors (see FIG. 2). The bimolecular FRET biosensors detect intermolecular interactions, whereas the unimolecular FRET biosensors detect conformational changes in molecules (see Aoki, K. and M. Matsuda. 2009. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nature Protocol 4:1623-1631, Giepmans, B. N., S. R. Adams, M. H. Ellisman, and R. Y. Tsien. 2006. The fluorescent toolbox for assessing protein location and function. Science 312:217-224, Jares-Erijman, E. A. and T. M. Jovin. Imaging molecular interactions in living cells by FRET microscopy. 2006. Curr. Opin. Chem. Biol. 10:409-416, and Kiyokawa, E., S. Hara, T. Nakamura, and M. Matsuda. 2006. Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells. Cancer Sci. 97:8-15).
Among them, there have been developed the unimolecular biosensors (unimolecular FRET biosensors) for quantifying small molecules such as ions, saccharides and lipids or for measuring activities of low molecular weight GTP-binding proteins or kinases (see Kiyokawa, E., S. Hara, T. Nakamura, and M. Matsuda. 2006. Fluorescence (Forster) resonance energy transfer imaging of oncogene activity in living cells. Cancer Sci. 97:8-15).
However, in order to create the biosensors, at least 3, often 4 or more protein domains are required to be connected. Unimolecular FRET biosensors having a satisfactory sensitivity usually cannot be achieved simply by connecting the protein domains to each other. That is, in order to create such unimolecular FRET biosensors, the following three factors should be taken into account: (i) an overlap of emission spectra of donor fluorescent proteins and absorption spectra of acceptor fluorescent proteins, (ii) a distance between the donor fluorescent proteins and the acceptor fluorescent proteins, and (iii) an orientation of emission moments of the donor fluorescent proteins and the absorption moments of acceptor fluorescent proteins. The fluorescent proteins may be fused with other proteins, which may apply stress to the fluorescent proteins to thereby cause misfoldings in the fluorescent proteins. As a result, the fluorescent proteins may form fluorophores inefficiently to thereby be nonfluorescent, which also should be taken into account. As described above, the FRET between the donor fluorescent proteins and the acceptor fluorescent proteins can be excellently achieved only when strict conditions are met. However, no requirement regarding, for example, an arrangement of the donor fluorescent proteins and the acceptor fluorescent proteins has been established. Therefore, for each unimolecular FRET biosensor to be created, optimizations have been performed by varying lengths of the protein domains or sequences of linkers connecting the domains, which needs a lot of trial and error and complex and advanced experiments. Accordingly, unimolecular FRET biosensors having a satisfactory sensitivity have been very hard to be developed.
Some linkers for connecting the domains have been reported such as a 9-amino acid linker consisting of glycine, serine and threonine (see Itoh, R. E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Activation of Rac and Cdc42 video-imaged by FRET-based single-molecule probes in the membrane of living cells. Mol. Cell. Biol. 22:6582-6591) and a 72-amino acid glycine linker (see Harvey, C. D., A. G. Ehrhardt, C. Cellurale, H. Zhong, R. Yasuda, R. J. Davis, and K. Svoboda. 2008. A genetically encoded fluorescent sensor of ERK activity. Proc. Natl. Acad. Sci. U.S.A. 105:19264-19269), which linkers have not been sufficiently optimized. Additionally, the above-described linkers could not be optimized in common with many types of unimolecular FRET biosensors.